Cerebellar cortex development in the weaver condition presents regional and age-dependent abnormalities without differences in Purkinje cells neurogenesis.

Ataxias are neurological disorders associated with the degeneration of Purkinje cells (PCs). Homozygous weaver mice (wv/wv) have been proposed as a model for hereditary cerebellar ataxia because they present motor abnormalities and PC loss. To ascertain the physiopathology of the weaver condition, the development of the cerebellar cortex lobes was examined at postnatal day (P): P8, P20 and P90. Three approaches were used: 1) quantitative determination of several cerebellar features; 2) qualitative evaluation of the developmental changes occurring in the cortical lobes; and 3) autoradiographic analyses of PC generation and placement. Our results revealed a reduction in the size of the wv/wv cerebellum as a whole, confirming previous results. However, as distinguished from these reports, we observed that quantified parameters contribute differently to the abnormal growth of the wv/wv cerebellar lobes. Qualitative analysis showed anomalies in wv/wv cerebellar cytoarchitecture, depending on the age and lobe analyzed. Such abnormalities included the presence of the external granular layer after P20 and, at P90, ectopic cells located in the molecular layer following several placement patterns. Finally, we obtained autoradiographic evidence that wild-type and wv/wv PCs presented similar neurogenetic timetables, as reported. However, the innovative character of this current work lies in the fact that the neurogenetic gradients of wv/wv PCs were not modified from P8 to P90. A tendency for the accumulation of late-formed PCs in the anterior and posterior lobes was found, whereas early-generated PCs were concentrated in the central and inferior lobes. These data suggested that wv/wv PCs may migrate properly to their final destinations. The extrapolation of our results to patients affected with cerebellar ataxias suggests that all cerebellar cortex lobes are affected with several age-dependent alterations in cytoarchitectonics. We also propose that PC loss may be regionally variable and not related to their neurogenetic timetables.

Regional differences in the vulnerability of substantia nigra dopaminergic neurons in weaver mice.

Vulnerability of midbrain dopaminergic (DA) neurons in the weaver mouse was studied at postnatal (P) days 8 and 90, in chosen coronal levels throughout the anteroposterior (AP) extent of the substantia nigra pars compacta (SNc). Wild-type (+/+) and homozygous weaver (wv/wv) mice used were the offspring of pregnant dams injected in several cases with tritiated thymidine on embryonic days 11-15. DA neurons were identified for their tyrosine hydroxylase immunoreactivity. Data reveal that at P8, the frequency of both +/+ and wv/wv late-generated DA cells increases from rostral to caudal SNc. No apparent DA-cell loss was observed at P8 in the mutant genotype, irrespective of the AP level considered. However, throughout the AP, there was a significant reduction in the number of these neurons at any level in 90-day-old weavers. Comparison of P8 and P90 +/+ SNc suggests that cell death is not a major aspect in the developmental regulation of normal DA neurons, although numerical cell depletion in the postnatal development of weaver SNc probably results from the amplification of a basal cell-death process, which affected all the coronal levels studied.

Purkinje cell age-distribution in fissures and in foliar crowns: a comparative study in the weaver cerebellum.

Generation and settling of Purkinje cells (PCs) are investigated in the weaver mouse cerebellum in order to determine possible relationships with the fissuration pattern. Tritiated thymidine was supplied to pregnant females at the time that these neurons were being produced. Autoradiography was then applied on brain sections obtained from control and weaver offspring at postnatal (P) day 90. This makes it possible to assess the differential survival of neurons born at distinct embryonic times on the basis of the proportion of labeled cells located at the two foliar compartments: fissures and foliar crowns. Our data show that throughout the surface contour of the vermal lobes, generative programs of PCs were close between wild type and homozygous weaver. Similar data were found in the lobules of the lateral hemisphere. On the other hand, the loss of PCs in weaver cerebella can be related to foliar concavities or convexities depending on the vermal lobe or the hemispheric lobule studied. Lastly, we have obtained evidence that late-generated PCs of both normal and mutant mice were preferentially located in fissures. These quantitative relationships lead us to propose a model in which the final distribution of PCs through the vermal contour would be coupled to two factors: the cortical fissuration patterning and a "time-sequential effect" of weaver mutation.

Generation and survival of midbrain dopaminergic neurons in weaver mice.

Generation and survival of midbrain dopaminergic (DA) neurons were investigated using tyrosine hydroxylase (TH) immunocytochemistry combined with tritiated thymidine autoradiography at appropriate anatomical levels throughout the anteroposterior (A/P) axes of the substantia nigra pars compacta (SNc) and the ventral tegmental area (VTA). The wild-type (+/+) and homozygous weaver (wv/wv) mice used here were the offspring of pregnant dams injected with the radioactive precursor when the mesencephalic neurons were being produced (gestational days 11-15). Data reveal that, at postnatal day 90, depletion of TH-stained cells in the wv/wv presented an A/P pattern of increasing severity and, therefore, the DA cells located in posterior parts of the SNc or the VTA appear to be more vulnerable than the settled anterior neurons. When the time of neuron origin is inferred for each level of these cell groups, it is found that the neurogenesis span is similar for both experimental groups, although significant deficits in the frequency of wv/wv late-generated neurons were observed in any level considered. On the other hand, it has been found that TH-positive neurons were settled along the extent of the SNc and the VTA following precise and differential neurogenetic gradients. Thus, the acute rostrocaudal increase in the proportion of late-generated neurons detected in both+/+DA-cell groups is disturbed in the weaver homozygotes due to the indicated A/P depletion.

The weaver gene expression affects neuronal generation patterns depending on age and encephalic region.

Cell generation and survival are investigated in three different neuronal populations of weaver mice: Purkinje and fastigial neurons in the cerebellum, and dopaminergic neurons in the substantia nigra pars compacta. Tritiated thymidine was supplied to pregnant females at the time that these neurons were being produced. Autoradiography was then applied on brain sections obtained from the control and weaver offspring at postnatal (P) day 8 and 90. This makes it possible to assess the differential survival of neurons that were born at distinct embryonic times on the basis of the proportion of labeled cells at two postnatal ages. When labeling profiles were measured at P8, the inferred time of origin was similar between +/+ and wv/wv genotypes for each neuronal population considered. The same occurred at P90 for Purkinje or fastigial neurons, but the labeling profiles of midbrain neurons were different between wild type and weaver homozygotes. There is already a substantial reduction in the number of Purkinje and fastigial cells at P8, but loss of dopaminergic neurons was only detected in 90-day-old weavers and, therefore, vulnerability is built into this midbrain neural system during its late postnatal development. Our results show that depletion of Purkinje and fastigial cells is random with respect to the time of their birth, whereas the weaver gene seems to be specifically targeting the late-generated dopaminergic neurons.

A combined immunohistochemical and autoradiographic method to detect midbrain dopaminergic neurons and determine their time of origin.

The present paper describes an efficient tyrosine hydroxylase immunohistochemical method combined with [3H]thymidine autoradiography to identify, in the same tissue section, substantia nigra pars compacta dopaminergic neurons and quantitatively determine their neurogenetic timetables both in control animals and in homozygous weaver mice. The experimental animals were the offspring of pregnant dams injected with [3H]thymidine on embryonic days 11-12, 12-13, 13-14 and 14-15. Preservation of tyrosine hydroxylase immunoreactivity, as well as the establishment of the best conditions for the [3H]thymidine autoradiography, were attained after many trials in which fixatives, buffers, antibody dilution and conditions for photographic emulsion, developer and fixer were tested. The proposed combined technique reveals that the detection of tyrosine hydroxylase positive neurons is accurate, unambiguous and has a low interassay variability. Moreover, it did not influence posterior [3H]thymidine autoradiography, which was highly reproducible and presented very low background. The method described here allows us to demonstrate that the neurogenetic timetables of midbrain dopaminergic neurons were different between control and homozygous weaver mice in the mesencephalic area studied.

Regional differences in the Purkinje cells settled pattern: a comparative autoradiographic study in control and homozygous weaver mice.

To determine whether Purkinje cells located in the vermis and the lateral hemispheres of weaver mice homozygotes are distributed according to precise neurogenetic gradients, [3H]thymidine autoradiography was applied on sections of homozygous weaver mice and normal controls on postnatal day 90. The experimental animals were the offspring of pregnant dams injected with [3H]thymidine on embryonic days 11-12, 12-13, 13-14, and 14-15. The results indicate that, at the level of the vermis, neurogenetic gradients were similar for wild-type and homozygous weaver in each lobe studied of the cerebellar cortex. The same was found for the lobulus simplex and for the ansiform and paramedian lobules when the lateral hemisphere was considered. In the vermis of both experimental groups, the anterior and inferior lobes have more late-generated Purkinje cells than the central and posterior lobes, while in the lateral hemisphere, the lobulus simplex and the ansiform lobule present more early generated Purkinje cells than the paramedian lobule. In weaver homozygotes, the most important deficit of Purkinje cells, in the region of the vermis, was observed in the central lobe; depletion was less observable in the anterior lobe and least observable in the posterior and inferior lobes. In the lateral hemispheres, the most important loss of Purkinje cells was observed in the paramedian lobule, followed by the lobulus simplex. The ansiform lobule presented values that showed no statistical difference between control and homozygous weaver. When Purkinje cells were registered in the entire sections, no significant differences were observed between the two experimental groups. This was due to a considerable volume of the weaver homozygote cerebellar tissue, which has no counterpart in the control mice, compensating for the neuronal loss observed in the other studied areas of the lateral hemisphere.

Regional differences in the stratified transitional field and the honeycomb matrix of the developing human cerebral cortex.

The neurons of the cerebral cortex originate in the proliferative neuroepithelium and settle in the cortical plate during embryonic development. Interposed between these two sites is a large transitional field. We have earlier demonstrated experimentally in rats with 3H-thymidine autoradiography that this transitional field is a stratified structure composed of discrete layers of migrating and sojourning cells, and fiber bands. Here we show that the different layers of the stratified transitional field are identifiable without experimental procedures in the developing human cerebral cortex and that there are conspicuous regional differences in its stratification. At the peak of its development, the stratified transitional field contains three fibrous bands in an inside-out order: the commissural fibers of the corpus callosum, the thalamocortical and corticofugal projection fibers, and the expanding white matter. There are regional differences in the thickness of these fibrous layers as well as in the number and configuration of the perikaryal layers. This preview focuses on laminar differences of the transitional fields of the agranular frontal lobe and the granular parietal and occipital lobes. At the latter sites, but not in the frontal lobe, there is a distinctive multi-layered band, the honeycomb matrix, where radially oriented fiber columns are sandwiched between two perikaryal sublayers and are separated from one another by radially oriented cells. We postulate that the radial fiber columns of the honeycomb matrix are composed of topographically organized thalamocortical fibers and that the unspecified young neurons acquire their enduring topographic identity by making selective contacts with tagged fibers here before they resume their radial or tangential migration to the cortical plate.